水稻叶面施硅调控籽粒镉积累的分子机理

At present, the situation of soil cadmium (Cd) pollution is becoming more and more serious owing to various anthropogenic activities. The prevention and control of cadmium pollution in rice has become a hot and difficult scientific issue in soil quality and food safety. It has been received widespread attention that soil application of silicon alleviates Cd toxicity and even reduce Cd accumulation in rice, however, there are few reports on the effect of foliar application of silicon on Cd uptake and transport and grain Cd accumulation in rice. In this project, firstly, we will use hydroponic cultivation, and the extreme-types of Cd accumulation in rice grain and the gene knockout mutants by CRISPR/Cas9 targeting technology obtained in our previous studies will be used as research materials. The qRT-PCR technique will be used to study the the responses of known Cd uptake and transport genes to foliar application of silicon. RNA-seq analysis will be used to analyze the changes of transcript levels before and after silicon application. The genetic networks and new genetic pathways related to silicon regulation of cadmium will be explored. Also new genes related to silicon regulation on Cd and their functions will be identified, meanwhile we will study the effect of silicon foliar application on the distribution of Cd and apoplastic transport. Then, using simulation pot experiment of polluted soil and natural cultivation in the field, the regulation effect and mechanism of silicon foliar application will be further validated. Finally, the mechanism of silicon foliar application on rice grain Cd accumulation will be analyzed synthetically, to provide a theoretical basis for silicon foliar application control of Cd. It is of great significance to reduce Cd accumulation in the edible part of rice and guarantee the quality and safety of the staple food in our country.

当前土壤镉污染形势日益严重，稻米镉污染防控已成为土壤质量与食物安全中的热点和难点科学问题。土壤施硅缓解水稻镉毒害甚至降低镉积累受到了广泛关注，但是水稻中关于叶面施硅影响镉吸收转运和籽粒镉积累的报道鲜见。本项目首先利用营养液水培，以前期获得的水稻籽粒镉积累极端类型品种、CRISPR/Cas9打靶技术定点敲除的基因突变体为材料，采用qRT-PCR技术研究已知的镉吸收转运重要基因对叶施硅的响应，并利用RNA-seq技术分析叶施硅前后转录组水平表达变化，发掘硅调控镉相关的基因网络和新遗传途径，探索新基因及其功能，同时研究叶面施硅对镉分布及质外体运输的影响，然后利用污染土盆栽模拟和大田自然栽培，对叶施硅调控效应和机理进一步验证探讨，最后综合理解叶施硅调控水稻籽粒镉的机制，为叶施硅控镉提供理论依据，对有效降低镉在可食部分积累，维系粮食质量安全具有十分重要意义。

Cd是水稻非必需的有毒元素。Si在缓解水稻Cd胁迫及减少Cd积累方面具有重要作用。本项目研究通过调查叶面喷Si与水稻生长、基因表达、Cd耐性/积累之间联系，解析施Si控Cd的作用与机制。研究表明，叶面喷Si不仅缓解水稻Cd胁迫、促进水稻生长，还可降低Cd从根部向地上部包括籽粒的转移，从而减少稻米Cd积累。灌浆期是叶面喷Si减少稻米Cd积累的最佳时期。在黄华占灌浆期喷Si，成熟期糙米Cd含量下降约65%。对灌浆期Cd胁迫下叶面喷Si的黄华占水稻进行转录组学分析，在剑叶、幼穗和节I中分别检测到328、209、298个Cd响应基因的表达施Si后回复到对照水平，而2075、1880、877个基因受施Si诱导差异表达。在剑叶、幼穗和节I中，分别检测到1221、678、643个Cd胁迫下Si特异响应基因。这些Si介导基因大多数表达上调，主要与转运与定位、生物合成与代谢、氧化还原、刺激响应、细胞壁形成等过程相关。尤其重要的是，Si介导剑叶和幼穗中大多数ABC转运蛋白上调表达、而剑叶和节I中大多数二价阳离子转运蛋白下调表达，还介导剑叶和幼穗中必需营养元素转运蛋白、碳水化合物和次生代谢物生物合成、细胞色素氧化酶活性等相关基因表达上调，这为叶面喷Si缓解水稻Cd胁迫、减少水稻Cd积累作用提供了直接的分子证据。转录组学分析还发现，水稻根系不仅存在对Cd、As胁迫共同响应的基因调控网络，还存在Cd、As胁迫下对施Si响应的共同生物学过程和代谢途径。本项目鉴定到两个Si介导的缓解Cd胁迫的基因，并对其中一个基因即硫素蛋白基因OsThi9的功能进行了深入研究。研究发现OsThi9蛋白可能螯合Cd离子，促进细胞内Cd外排并结合到细胞壁上，从而减少Cd导致细胞内氧化损伤，进而提高水稻植株Cd耐性、促进植物生长，并可能减少Cd积累。这些结果为Cd污染农田叶面喷Si调控水稻籽粒Cd积累、低Cd水稻分子育种等提供了理论依据和候选靶基因。本项目研究已发表SCI论文4篇、申请专利1项（已公开）、已培养毕业硕士研究生4人。